In the pursuit of novel therapeutic agents: synthesis, anticancer evaluation, and physicochemical insights of novel pyrimidine-based 2-aminobenzothiazole derivatives

Cancer remains a worldwide healthcare undertaking, demanding continual innovation in anticancer drug development due to frequent drug resistance and adverse effects associated with existing therapies. The benzothiazole compounds, particularly 2-aminobenzothiazole derivatives, have attracted interest for their versatility in generating novel anticancer agents. This study explores the synthesis, and anticancer evaluation of new pyrimidine-based 2-aminobenzothiazole derivatives. A range of synthetic methods have been developed based on the reaction of 2-benzothaizolyl guanidine with various reagents such as α,β-unsaturated carbonyl, 2-cyano-three-(dimethylamino)-N-acrylamide, β-diketones, β-keto esters, and S,S ketene dithioacetals. Human tumour cell lines such as HepG2, HCT116, and MCF7 were used in in vitro cytotoxicity studies, and the results showed that several of the synthesized compounds were more potent than the standard drug, 5-fluorouracil, in terms of cell viability% with low IC50. Furthermore, the computed drug likeness and ADMET properties of the most potent synthesized compounds suggest their potential as promising candidates for further development, with favorable bioavailability and pharmacokinetic profiles.


Introduction
Cancer has emerged as a signicant healthcare concern worldwide, with a rising number of cases over time.To address this issue, numerous anticancer drugs have been approved and are currently in clinical use.However, the challenges of drug resistance and adverse effects persist, creating a continual need for innovative, potent, and safe candidates for cancer therapy.In recent decades, researchers have explored and documented various heterocyclic ring-based derivatives in the literature.Notably, benzothiazole scaffold-based compounds have proven to be versatile rings for the development of novel and safe anticancer candidates.The scaffold of 2-aminobenzothiazole has undergone extensive exploration to create diverse analogues demonstrating remarkable biological activity against various targets.Notably, several therapeutic agents incorporating this framework have received clinical approval.One such example is riluzole, a vital drug based on 2-aminobenzothiazole, employed in treating amyotrophic lateral sclerosis, a severe neurodegenerative disorder 1 (Fig. 1).Many studies have indicated its promising anti-tumor effects on various human solid cancer cell lines. 2Another signicant compound is tioxidazole, an anthelmintic drug designed for the treatment of parasitic infections 3 (Fig. 1).Additionally, frentizole, depicted in Fig. 1, serves as a non-toxic antiviral and immune suppressive agent utilized in clinical settings for conditions such as rheumatoid arthritis and systemic lupus erythematosus. 4 In recent developments, 2-aminobenzothiazole derivatives have emerged as novel antineoplastic agents, showcasing a diverse range of protein targets, including tyrosine kinases such as EGFR, CSF1R, VEGFR-2, MET, and FAK, serine/ threonine kinases such as Aurora, CK, CDK, DYRK2, and RAF, mutant p53 protein, BCL-XL, PI3K kinase, HSP90, NSD1, HDAC, LSD1, DNA topoisomerases, FTO, mPGES-1, hCA IX/XII, SCD, and CXCR receptor. 5Concurrently, 2-aminobenzothiazole stands as a prominently featured scaffold in medicinal chemistry, prevalent in bioactive molecules, particularly those pertaining to cancer agents-exemplied by compounds A, B, and C, [6][7][8] Fig. 2. 2-Aminobenzothiazoles with a pyrimidine base, in particular, have demonstrated noteworthy anticancer activities against various cell lines and enzymes.For example, a series of cyano and amidinobenzothiazole-substituted anilins were synthesized and assessed for their antiproliferative effects on various tumor cell lines, such as Hep-2, MCF-7, HeLa, MiaPaCa-2, SW 620, and H 460. Notably, the pyrimidine-based carbonitrile benzothiazole derivative D exhibited potency against all cancer cell lines studied, 9 Fig. 2. Additionally, derivatives of 2aminobenzothiazole, incorporating both isoxazole and pyrimidine rings, were synthesized and evaluated for their anticancer activity using the MTT assay across diverse cell lines, including A549, Colo205, MCF-7, and U937.Among them, compound E demonstrated notable anticancer efficacy against Colo205 and U937, exhibiting a potential IC 50 value in comparison to the standard drug etoposide, 10 Fig. 2.Moreover, compounds F and G displayed notable efficacy against three leukemia cell lines and protein tyrosine kinase (PTK), demonstrating inhibitory concentrations of 0.131 mM and 0.161 mM, respectively. 11arious methods have been utilized for the synthesis of diverse derivatives of pyrimidine-based 2-aminobenzothiazole.One approach involves the nucleophilic substitution reaction of commercially available 2,4-dichloro-5-methylpyrimidine with 2aminobenzothiazole at the C-4 position of the pyrimidine ring in the presence of sodium hydroxide (NaOH) at room temperature, yielding N-(2-chloro-5-methylpyrimidin-4-yl)benzo [d]thiazol-2-amine. 12Another method involves the reaction of 2benzothaizolyl guanidine with various molecules.The synthesis of 2-benzothaizolyl guanidine involves treating 2-aminobenzothiazole with S-methyl isothiourea or reacting cyano guanidine with o-aminothiophenol in an acidic medium. 11,13he resultant 2-benzothaizolyl guanidine reacts with substituted benzaldehydes and ethyl acetoacetate, methyl acetoacetate, or ethyl cyanoacetate, following Biginelli's method with modications, to yield pyrimidine-based 2-aminobenzothiazole derivatives. 11Additionally, 2-benzothaizolyl guanidine, when treated with methyl or ethyl acetoacetate in the presence of an excess of trimethylorthoacetate (TMOA) under nitrogen, results in another series of derivatives of pyrimidinebased 2-aminobenzothiazole, dependent on the involved 1,2diketones. 146][17][18] Triuoromethyl-substituted N-(pyrimidin-2-yl) benzo[d]thiazol-2-amines are prepared through the cyclocondensation reaction of 2-benzothaizolyl guanidine with 4alkoxy-4-alkyl(aryl/heteroaryl)-1,1,1-triuoroalk-3-en-2-ones or 2,2,2-triuoro-1-(2-methoxycyclohexen-1-en-1-yl)ethanone. 19oreover, a series of pyrimidine-base 2-aminobenzothiazoles is obtained by reacting 2-benzothaizolyl guanidine with ethyl 2butylacetoacetate, diethyl ethoxymethylenemalonate, ethyl ethoxymethylenecyanoacetate, and ethoxymethylenemalononitrile in a basic medium. 20n our previous investigations, we undertook the design and synthesis of a series of innovative benzothiazole derivatives in conjunction with pyrimidine, 21 pyridine, 22,23 purine analogues, 24 or thiophene ring. 25These compounds were evaluated for their antimicrobial, antiviral, and/or anticancer activities. 26Recognizing the key role of 2-aminobenzothiazole as a promising anticancer agent, our exploration was encouraged to create new derivatives of 2-aminobenzothiazole, specically in collaboration with the pyrimidine ring.This strategic modication aimed to further enhance the structural features and optimize the potency of the compounds.In this manuscript, we present our comprehensive investigation encompassing the synthesis, anticancer evaluation, and molecular docking studies of the newly designed pyrimidine-based 2-aminobenzothiazole derivatives.

Chemistry
To effectuate the synthesis of our designated novel pyrimidinebase 2-aminobenzothiazole derivatives, the initiation of the synthetic pathway involved the utilization of 2-benzothiazolyl guanidine 3 as the foundational precursor.The synthesis of 2benzothiazolyl guanidine was accomplished through a straightforward method.This method involved the condensation reaction between o-aminothiophenol 1 and cyanoguanidine 2, facilitated by an acidic medium and heating at 80 °C. 13his method was chosen based on the readily available starting materials and the overall efficiency and scalability of the process.Several a,b-unsaturated carbonyl compounds 4a-d, as illustrated in Scheme 1, underwent a reaction with 2-benzothiazolyl guanidine 3 to produce 2-aminobenzothiazoyl pyrimidine 7a-d, featuring a cyano group at position 5.The Utilizing symmetrical and unsymmetrical diketones, namely 14a and 14b, respectively, yielded a single cyclization product based on NMR spectra.Additionally, reactions involving benzothiazolyl guanidine 3 and triethyl orthoformate with b-keto ester 14c, potentially resulting in two intermediates, produced a singular cyclization product, 2-aminobenzothiazolyl ethoxycarbonylpyrimidine 15c, without the formation of hydroxyacylpyrimidine.The existence of the ester group in compound 15c was conrmed by observing a triplet and quartet of the ethoxy group at chemical shis of d 1.07 and 4.14 ppm, respectively, in its 1 H NMR spectra.
The Aldol condensation reaction was carried out on compound 15a with aromatic aldehyde derivatives to produce the corresponding chalcones, which feature an a,b-unsaturated carbonyl system.Chalcones, a subgroup of avonoids, were synthesized in this study by reacting 2-aminobenzothiazolyl acylpyrimidine 15a with substituted aromatic aldehydes 16a-d in a basic medium, using ethanol as the solvent.This process led to the formation of 2-aminobenzothiazolyl pyrimidine-linked chalcones 17a-d, Scheme 3. The structure of the newly synthesized compounds was determined through IR and NMR spectroscopy.For instance, the IR spectrum of 17a revealed an absorption band at 1663 cm −1 , indicating the presence of a C]O group (conjugated ketone).In the 1 H NMR spectrum of 17b, two doublet signals at d 7.58 and 7.82 ppm, with a coupling constant of 15.5 Hz, provided evidence of the E conguration of the produced chalcones.
Following a 6 hours reux in the presence of excess hydrazine hydrate, 2-aminobenzothiazolyl ethoxycarbonylpyrimidine  Moreover, in the presence of potassium hydroxide, 2-benzothiazolyl guanidine 3 was reacted with 2-benzothiazolyl enaminoacrylonitrile 23. 28As shown in Scheme 4, this reaction produced N 2 ,5-bisbenzothiazolyl pyrimidine 24.Compound 24's structure was determined by thoroughly analyzing its IR and NMR spectra.The IR spectra showed characteristic absorption bands for the NH 2 and NH groups, which were situated at around 3463 and 3266 cm −1 , respectively.Four protons of the two benzothiazole rings were attributed to four doublet signals in the 1   the elimination of NH(CH 3 ) 2 .Subsequently, intramolecular cyclization occurred through the addition of the amino group to the cyano group, ultimately yielding the pyrimidine derivative 24.

In vitro cytotoxic activity
The cytotoxic impact of novel pyrimidine-based 2-aminobenzothiazole derivatives 7a-d, 13a-c, 15a-c, 17a-d, 18, 20, 22, 24 was assessed utilizing the standard 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) bioassay.In the initial phase, the synthesized compounds underwent screening for their anticancer activities against three human tumor cell lines, HepG2, HCT116, and MCF7, at a singular concentration 100 mmol mL −1 .Cell viability percentages were utilized as the metric for assessing outcomes, and comparisons were made with 5-uorouracil, chosen as the standard positive control drug.5-Fluorouracil is a chemotherapeutic agent employed in the treatment of diverse malignancies including gastric adenocarcinoma, pancreatic adenocarcinoma, breast carcinoma, and colorectal adenocarcinoma. 29The results are detailed in Tables 1 and S1  In the second phase and based on the screening results, the IC 50 , which represents the compound concentrations required to produce a 50% inhibition of cell growth aer 72 h of incubation, was measured for the most potent compounds.Specically, compounds 7c, 13b, 17d, and 18 were evaluated for the HepG2 cell line.For the HCT116 cell line, compounds 7b, 13c, and 15c were assessed, and compound 24 was tested for the MCF7 cell line.The IC 50 values were determined through analysis of the concentration-inhibition response curve, Fig. 3  0.01 mmol mL −1 .The second most potent compound against HepG2 is compound 18 with IC 50 0.53 ± 0.05 mmol mL −1 followed by compound 13b with IC 50 of 0.56 ± 0.03 mmol mL −1 .Notably, three of the newly synthesized compounds, 17d, 18 and 13b, demonstrated higher potency, based on the resulting IC 50 data, compared to 5-uorouracil, which has an IC 50 of 1.03 mmol mL −1 . 30Surprisingly, compound 15c displayed an IC 50 of 0.02 ± 0.001 mmol mL −1 , indicating superior efficacy against HCT116 compared to 5-uorouracil, which exhibited an IC 50 of 9 ± 1.7 mmol mL −1 . 31Compound 15c not only demonstrated heightened potency relative to 5-uorouracil but also exhibited notable efficacy alongside compounds 7b and 13c, which displayed IC 50 values of 2.95 ± 0.26 and 1.033 ± 0.06, respectively.Additionally, compound 24 exhibited IC 50 value, 1.485 ± 0.15 mmol mL −1 , lower than the IC 50 value of 5-ouracel, 7.12 mmol mL −1 , against MCF7. 32These results suggest that the investigated compounds exhibit potential as robust anticancer agents.
The ndings from this study reveal that the inclusion of halogen groups, specically F and Cl, on the aryl group bonded with pyrimidine-based 2-aminobenzothiazole 7a-d resulted in an increase of compound activity.Additionally, the incorporation of CO 2 Et, as observed in compound 15c, enhanced the potency of the compound in comparison to its analogs, 15a and 15b, containing COCH 3 and COPh, respectively.In the context of pyrimidine-based 2-aminobenzothiazole 17a-d, the presence of a methoxy group was found to amplify the compound's potency more signicantly than those possessing halogen substituents.Notably, compounds featuring SCH 3 exhibited the lowest activity levels across the three tested cell lines.

Drug likeness, and physicochemical-pharmacokinetic/ ADMET properties
To assess the potential of the synthesized compounds as drug candidates, various parameters including drug likeness, adherence to specic rules, and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties were computed using Molso soware and the SwissADME program. 33Poor oral bioavailability in drug discovery is oen    associated with characteristics such as more than ve hydrogen bond donors, ten hydrogen bond acceptors, a molecular weight exceeding 500 g mol −1 , and a calculated log P greater than 5. Analysis of the results in Table 5 indicates that all potent compounds exhibited only one or no violation in these criteria.Specically, none of the compounds surpassed the normal range for the number of hydrogen bond donors, number of hydrogen bond acceptors, and log P.Moreover, all compounds demonstrated a drug-likeness score within the range of 0.05 to 0.84.Further examination revealed that the molecular weight and topological polar surface area (TPSA) of compounds 7b, 7c, 13c, 15c, 17d, and 18 did not exceed the standard limits of 500 g mol −1 and TPSA of 140 Å 2 , except for compounds 13b and 24, which exhibited slightly higher TPSA values of 143.29 and 146.09Å 2 , respectively.
Furthermore, the investigation into blood-brain barrier (BBB) permeability, gastrointestinal (GI) absorption, and bioavailability of the synthesized compounds was conducted using the SwissADME program, Table 6.The results presented in Table 6 indicate that all potent synthesized compounds exhibit no blood-brain barrier permeability, suggesting their inability to traverse the BBB.Conversely, compounds such as 7b, 7c, 13b, 13c, and 24 demonstrate low GI absorption, revealing of favorable absorption in the human intestine.In contrast, compounds 15c, 17d, and 18 exhibit high GI absorption.Furthermore, all potent compounds boast a bioavailability score of 0.55, implying favorable pharmacokinetic properties.

Docking study
Protein tyrosine kinases (PTKs) play a crucial role in regulating the proliferation, differentiation, and signaling processes within immune system cells.They are broadly categorized into transmembrane receptor-linked kinases and cytoplasmic kinases. 34Abnormal signaling from both receptor tyrosine kinases and intracellular tyrosine kinases can contribute to various diseases, particularly cancer, including non-small cell lung cancer, chronic myeloid leukemia, and gastrointestinal stromal tumors. 35Due to its signicant involvement in cancer etiology, the PTK receptor has been a subject of considerable attention for a considerable period.Aminobenzazoles, such as aminobenzothiazole, aminobenzoxazoles, and benzimidazoles, when paired with pyrimidines, have emerged as potential inhibitors of PTK. 11Given the structural similarity between our synthesized compounds and previously proven potent compounds against protein tyrosine kinases, 11 we conducted molecular docking studies on the PTK receptor (PDB ID: 2GQG) to elucidate the interactions of the most effective compounds with the PTK binding site.
The validation of the docking study involved placing the cocrystallized ligand (1N1) inside the active site aer extraction from the respective receptor, as illustrated in Fig. 6.The docking of the cocrystallized ligand 1N1 yielded a root mean square deviation value of 1.1559 and binding energy −9.2907 kcal mol −1 , Table 7.The results indicated that 1N1 formed one H-bond acceptor with Met318, two H-bond donors with Met318 and Thr315, and one arene-H interaction with Leu248.Fig. 6A and B depicts the various types of interactions between the ligand 1N1 and the PTK active site.
The top-ranked poses of the most active compounds, 7b, 7c, 13b, 13c, 15c, 17d, 18 and 24, within the active site of PTK are summarized in Fig. 7A-H.Notably, the docking analysis revealed that all compounds t inside the active site.It was observed that all compounds formed a hydrogen donor bond with Met318, except for 15c.Among these compounds, 15c, 17d, 18, and 24 exhibited binding energies closer to the cocrystallized ligand 1N1, with values of −7.7288, −7.3209, −7.6805, and −7.5790 kcal mol −1 , respectively, Table 7.Despite compound 13c having a binding energy of −6.9625 kcal mol −1 , it demonstrated four interactions with the active site, including one arene-H interaction with Leu248 and three H-bond donors with Met318 and Thr319.Observations revealed that all compounds, particularly 13c, 15c, 17d, 18, and 24, demonstrated promising interactions with the active site of PTK.However, it is important to note that we were unable to conduct an in vitro study due to the unavailability of the required kit.

Conclusion
In conclusion, our investigation has led to synthesis, and evaluation of new derivatives of pyrimidine-based 2-aminobenzothiazole as potential anticancer agents.The study has successfully elucidated and developed various synthetic routes, leading to a high yield of pyrimidine-based 2-aminobenzothiazole compounds.The conrmation of the desired compounds was achieved through comprehensive analytical and spectral analyses.The results of in vitro cytotoxicity studies revealed that several synthesized compounds displayed potent activities against human tumor cell lines, including HepG2, HCT116, and MCF7.Compounds 17d, 18, and 13b demonstrated notable efficacy, with IC 50 values lower than that of the reference drug, 5-uorouracil, in the case of HepG2.Moreover, compound 15c exhibited superior potency compared to 5-uorouracil against HCT116.The physicochemical and pharmacokinetic properties of the synthesized compounds were assessed, and the majority adhered to drug-likeness criteria, indicating their potential as drug candidates.Furthermore, based on the docking study and a comparative analysis with previously similar compounds, the identied potent compounds exhibit promising potential as inhibitors of PTK.The overall ndings suggest that these newly synthesized derivatives of pyrimidinebased 2-aminobenzothiazole hold promise as novel anticancer agents, warranting further exploration and optimization for potential clinical applications.

Chemistry
An SMP3 melting point equipment was used to determine melting points.The 1 H NMR spectra (400 MHz) were obtained at Ain Shams University in Cairo, Egypt, using a Bruker Advance (III)-400 MHz Spectrometer.The 13 C NMR spectra (125 MHz) were collected using a Bruker Advance (III)-600 MHz Spectrometer at Helwan University's Central Laboratory, Hub of 4.2.Anticancer evaluation 4.2.1 Preparation of pyrimidine derivatives provided compound.A 100 mmol mL −1 stock solution was created by reconstituting the dried extract in an appropriate volume of DMSO, based on the molecular weight of each compound, followed by 5 seconds of sonication.This stock solution was aliquoted and stored at −20 °C until needed.Final test compound concentrations for all experiments were prepared by diluting the stock with the medium.The control cells received the carrier solvent (0.1% DMSO).
4.2.2.2 Assessment of cell viability by cell proliferation assay (MTT) 48 hours aer culture.One day before conducting the experiment, the cancer cells were seeded in 96-well culture plate.8 × 10 3 cells per well were seeded in 200 mL of DMEM, supplemented with 10% FBS and 1% of penicillin G sodium (10.000UI), streptomycin (10 mg) and amphotericin B (25 mg) (PSA) (Gibco, Thermosientic, Germany).Culture plates were incubated at 37 °C in an atmosphere of 5% CO 2 for 24 hours for attach of cells.On the next day, a constant concentration of 100 mmol mL −1 , was prepared for each compound and used for treatment of cells.In addition, the carrier solvent (0.1% DMSO) was used for control cells.The 5-Fluorouracil was used as positive control for the three cancer cell lines with a concentration 140.0 mmol mL −1 , 36 1.1 mmol mL −1 , 37 and 125 mg mL −1 , 38 on MCF7, HepG2, and HCT116; respectively.Cells were maintained at 37 °C in an atmosphere of 5% CO 2 for 48 hours.At the end of incubation, the cell proliferation assay was performed using the Vybrant® MTT Cell Proliferation Assay Kit, cat no: M6494 (Thermo Fisher, Germany).100 mL of media was removed from and replaced by fresh media.Twenty mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (1 mg mL −1 ), Invitrogen, Thermo-Scientic, Germany was added to each well and the plates were incubated at 37 °C and 5% CO 2 for four hours.Finally, the MTT solution was removed and 100 mL of sodium dodecyl sulphate with hydrochloric acid (SDS-HCL) was added to the wells.Cell viability was determined by measuring the optical density at 570 nm on a spectrophotometer (ELx 800; Bio-Tek Instruments Inc., Winooski, VT, USA).
4.2.3Determination of the half maximal cytotoxic effect (IC 50 ) half maximal cytotoxic effect (IC 50 ) of pyrimidine derivatives on three cancer cell lines.One day before conducting the experiment, the cancer cells were seeded in 96-well culture plate.8 × 10 3 cells per well of cells were seeded in 200 mL of Dulbecco's Modied Eagle Medium (DMEM) (Gibco, Thermosientic, Germany) with high glucose (4.5 g L −1 ), L-glutamine and sodium pyruvate, containing 10% fetal bovine serum (FBS) (Gibco, Thermosientic, Germany) and 1% of penicillin G sodium (10.000UI), streptomycin (10 mg) and amphotericin B (25 mg) (PSA) (Gibco, Thermosientic, Germany).Culture plates were incubated at 37 °C in an atmosphere of 5% CO 2 for 24 hours to reach the 70% conuence.On the next day, a serial concentration of each tested compound was performed "100 mmol mL −1 , 10 mmol mL −1 , 1.0 mmol mL −1 , 0.1 mmol mL −1 , and 0.01 mmol mL −1 " were prepared and used for treatment of cells.In addition, the carrier solvent (0.1% DMSO) was used for control cells.
The treated cancer cells were incubated at 37 °C in an atmosphere of 5% CO 2 for 72 hours, then the cell viability was tested by MTT assay and the IC 50 was calculated.At the end of incubation time, the cell cytotoxicity assay was performed using the Vybrant® MTT Cell Proliferation Assay Kit, cat no: M6494 (Thermo Fisher, Germany).100 mL of media was removed and replaced by new media.Twenty mL of 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (1 mg mL −1 ) (Invitrogen, ThermoScientic, Germany) was added to each well and the plates were incubated at 37 °C and 5% CO 2 for four hours.Finally, the MTT solution was removed and 100 mL of sodium dodecyl sulphate with hydrochloric acid (SDS-HCL) was added to the wells.Cell viability was determined by measuring the optical density at 570 nm on a spectrophotometer (ELx 800; Bio-Tek Instruments Inc., Winooski, VT, USA).
4.2.4Calculation of IC 50 of pyrimidine derivatives on three cancer cell lines.At the end of each time interval, the cell proliferation assay was conducted, and the % of viability was determined which represent the cytotoxic effect of serial doses of each compound.The XY curve was plotted to illustrate the relation between the log dose of (inhibitor) versus the normalized response.The best t point was determined by linear regression analysis.Calculation of half maximal stimulatory concentration (IC 50 ).The IC 50 was calculated using the Graph-Pad prism soware, Prism 9, version 9.1.0(221).The determination of IC 50 for each group was calculated based on concentration-response curves of analyzed cellular metabolic activity, which were normalized to untreated cells.

Drug likeness predictions and physicochemicalpharmacokinetic/ADMET properties
Drug-likeness, a qualitative concept integral to drug design, plays a key role in predicting drug-like properties.Essential factors such as solubility, permeability, transporter effects, and metabolic stability signicantly inuence the success of drug candidates by impacting oral bioavailability, toxicity, metabolism, clearance, and in vitro pharmacology.To assess the drug-likeness of the synthesized compounds, ve distinct lters-Lipinski, 39 Veber, 40 Muegge, 41 Ghose, 42 and Egan 43 rules-were employed, along with considerations for bioavailability.Furthermore, drug-likeness scores were determined using both the Molso soware and the SwissADME program.

Molecular docking study
The molecular studies were conducted using the Molecular Operating Environment (MOE 2014).Ligand molecules were drawn using the builder molecule, and their energy was minimized.The minimization process continued until an rmsd gradient of 0.01 kcal mol −1 was achieved, employing the MMFF94X force eld, with automatic calculation of partial charges.Docking simulations utilized the crystal structure of the PTK receptor in complex with 1N1 from the Protein Data Bank (PDB ID: 2GQG).Ligands bound to the structure were excluded, and the MOE protonate 3D application was employed to add missing hydrogens and accurately assign ionization states.For the generation of the active site, the MOE-Alpha site nder was utilized, and the obtained alpha spheres were employed to create dummy atoms.Subsequently, ligands were docked into the active sites using MOE-Dock.The ranking of optimized poses was determined using GBVI/WSA DG freeenergy estimates, and the docking poses underwent visual examination.The nal step involved investigating interactions with binding pocket residues.
15c was entirely utilized to generate the corresponding hydrazide 18. Upon cooling, a white solid with a melting point of 294-295 °C was isolated.The IR spectra exhibited a band at 1628 cm −1 , indicative of the amide group's carbonyl (CO) functionality.The 1 H NMR analysis conrmed the absence of the ethoxycarbonyl group in the initial compound 15c, and revealed the presence of NH 2 at d 4.51 ppm and NH at d 9.66 ppm in the produced hydrazide group.Furthermore, our investigation was extended to encompass the reaction of benzothiazolyl guanidine 3 with S,S ketene dithioacetals, such as 2-(bis-(methylthio)methylene)malononitrile 19 and ethyl 2-cyano-3,3-bis(methylthio)acrylate 21, as depicted in Scheme 4. The reaction was conducted using KOH in dioxane, yielding the respective 2-aminobenzothiazol-4methylthio pyrimidine, 20, and 22.The suggested synthetic route for the target compounds entails the incorporation of the amino group of 3 into the ylidene bond in 19 and 21.Subsequently, this is followed by either eliminating an ethanol molecule when utilizing compound 21 or adding to the cyano group when utilizing compound 19.Finally, the cyclization occurs via the addition of the NH group to the cyano group.Elemental analysis and spectral data conrmed the proposed structures of compounds 20 and 22.The IR spectra clearly indicated the presence of NH and CN groups in both 20 and 22, as evidenced by absorption bands at 3378-3379 and 2198-2208 cm −1 , respectively.The 1 H NMR of 20 and 22 revealed a singlet signal at d 2.69-2.72 ppm, conrming the presence of SCH 3 protons.In the case of compound 20, a broad signal at d 7.70 ppm affirmed the existence of NH 2 groups.Additionally, the 13 C NMR spectra of compounds 20 and 22 displayed signals at d 40.4-40.5 ppm for the SCH 3 group and signals at d 115.6-118.4ppm for the CN group.
-S6 (ESI) † In light of the obtained results, four compounds 7c, 13b, 17d, and 18 demonstrated strong efficacy against the HepG2 cell line, exhibiting cell viability percentages of 61.29, 68.18, 61.04, and 66.85, respectively, in comparison to the standard drug with a cell viability percentage of 64.41.Another compounds showed moderate activities against HepG2 cell line such as 7b, 13a, 15c, and 17b with cell viability percent of 72.70, 72.35, 72.69 and 76.02.Additionally, three compounds 7b, 13c, and 15c exhibited slightly strong efficacy against the HCT116 cell line, displaying cell viability percentages of 70.62, 67.11, and 65.68, respectively, in contrast to the standard drug with a cell viability percentage of 55.96.Furthermore, a singular compound 24 demonstrated efficacy against the MCF7 cell line, with a cell viability percentage of 69.98, compared to the standard drug with a cell viability percentage of 62.76.

Fig. 3
Fig. 3 Nonlinear regression curve illustrating the log dose of pyrimidine derivatives 7c, 13b, 17d and 18 versus the normalized response in HepG2 cells after treatment with serial concentrations in DMEM for 72 hours.

- 5 .Fig. 4
Fig. 4 Nonlinear regression curve illustrating the log dose of pyrimidine derivatives 7b, 13c and 15c versus the normalized response in HCT116 cells after treatment with serial concentrations in DMEM for 72 hours.

Fig. 5
Fig. 5 Nonlinear regression curve illustrating the log dose of pyrimidine derivative 24 versus the normalized response in MCF7 cells after treatment with serial concentrations in DMEM for 72 hours.

Fig. 6
Fig. 6 Docking poses of 1N1 ligand inside PTK active site.(A) 2D interaction of 1N1 ligand with active site.(B) 3D docking of 1N1 ligand for validation.

Table 1
Viability% of synthesized compounds against HepG2, HCT116 and MCF7 cell lines

Table 3
Determination of IC 50 of compounds 7b, 13c and 15c on HCT116 cells

Table 4
Determination of IC 50 of compound 24 on MCF7 cells

Table 5
Drug likeness predictions and physicochemical-pharmacokinetic/ADMET properties of the most active compounds a Number of hydrogen bond acceptors.b Number of hydrogen bond donors.c Lipophilicity. d Topological polar surface area.

Table 6
Predicted ADMET properties of the tested compounds

Table 7
Molecular docking free binding energy and bond interactions estimate to PTK receptor © 2024 The Author(s).Published by the Royal Society of Chemistry RSC Adv., 2024, 14, 16332-16348 | 16341 Paper RSC Advances